Project Summary The ability to engineer living cells as intelligent therapeutic agents is poised to transform current cancer treatment paradigms. Based on the inherent growth specificity of some natural and exogenous bacteria to solid tumors, microbes have been explored as programmed vehicles to deliver therapeutics to tumor environments. However, a universal challenge for developing this next-generation living therapy is the lack of tools to study the dynami- cally interacting population of bacteria and cancer cells. Consequently, the vast majority of the past studies have relied on animal models that only test a handful of therapeutic candidates and provide limited spatiotemporal information. To address this challenge, I have recently developed a 3D multicellular coculture platform that ena- bles high-throughput characterization of bacteria in tumor spheroids, assessing dynamics of multicellular inter- actions and predicting therapeutic efficacy in vivo. In this proposal, I will leverage the 3D coculture technologies and synthetic biology tools to construct novel bacterial systems that sense and express therapeutics specifically in tumors. In the F99 phase, I will engineer tumor-homing bacteria to dynamically regulate production of cytotoxic molecules in response to tumor shrinkage. Specifically, as the cancer cell death increases the level of oxygen in the tumor core, I will engineer a bacterial biosensor circuit to monitor this change in the tumor microenvironment. By detecting rises in oxygen levels, which indicate extensive therapeutic progression, this gene circuit will reduce the therapeutic level produced, minimizing off-target toxicity. In the K00 phase, I will repurpose tumor-homing bacteria to target and eradicate oncogenic F. nucleatum in colorectal cancer. I will apply the 3D coculture platform to screen anti-F. nucleatum peptides delivered by bacteria to tumor spheroids. Orthotropic mouse cancer models will be used to assess the F. nucleatum elimination, effect on commensal microbiota, and cancer progression. Collectively, the proposed work will utilize a novel engineering framework to develop effective bacterial cancer therapies towards clinical translation.